| 研究課題/領域番号 |
19F19782
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| 研究機関 | 国立研究開発法人理化学研究所 |
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研究代表者 |
白須 賢 国立研究開発法人理化学研究所, 環境資源科学研究センター, グループディレクター (20425630)
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| 研究分担者 |
FISHMAN MAX 国立研究開発法人理化学研究所, 環境資源科学研究センター, 外国人特別研究員
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| 研究期間 (年度) |
2019-11-08 – 2022-03-31
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| キーワード | parasitic plants / calcium signaling / phosphoproteomics / DMBQ / haustorium |
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| 研究実績の概要 |
I received and analyzed phosphoproteomics data from Phtheirospermum japonicum roots treated with 10 uM DMBQ or a DMSO control. Among the differentially phosphorylated proteins were two receptor-like kinases (RLKs) that are homologous to the RLK in Ararbidopsis thaliana that is necessary for DMBQ-induced Ca2+ response. From these data I selected 11 putative gene candidates, including the two previously mentioned RLKs, for involvement in the primary haustorium signaling pathway and transformed P. japonicum with RNA interference (RNAi) constructs for each of those genes. These RNAi constructs include the Ca2+ signaling reporter R-GECO. I have currently performed preliminary tests on the effect of these RNAi constructs on Ca2+ signaling and haustorium development and the results are still pending.
Lastly, Target of rapamycin (TOR) kinase was one of the proteins differentially phosphorylated following DMBQ treatment. It has a high potential for being involved in haustorium formation based as several proteins downstream of TOR kinase are differentially phosphorylated following DMBQ treatment. I have performed chemical genetics using selective inhibitors for TOR kinase and found that they inhibit haustorium formation following DMBQ treatment. I am currently determining whether an RNAi construct can knockdown expression of TOR and reduce haustorium formation and investigating the expression localization of this gene in P. japonicum.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
I feel that I have progressed on the research plan I made last year. For that research plan, I had the goal of sending samples for phosphoproteomic analysis, analyzing the data, and then producing P. japonicum RNAi knockdown transformants to analyze the role of select genes in haustorium induction. On top of that, I have likely identified one gene already from the phosphoproteomics data that is involved in haustorium induction and am preparing to test the role of the phosphosite in its function. I have also collected root tip samples that are ready to be sent to a collaborator for targeted phosphoproteomic anlaysis.
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| 今後の研究の推進方策 |
Gene candidates of interest from Phtheirospermum japonicum phosphoproteomics data have been identified. Gene silencing constructs for these candidates will be further tested to determine if they are involved in haustorium formation and calcium signaling in P. japonicum. The DMBQ-sensitive phosphosites on identified candidates that were found to influence haustorium formation will then be confirmed using targeted phosphoproteomics. Homologous genes to the positive candidates will be identified in Arabidopsis thaliana and mutant lines of those genes will be crossed with A. thaliana expressing aequorin. The resulting individuals will be investigated for changes to DMBQ-related calcium signaling in A. thaliana. If any of these mutants show a difference in DMBQ-related calcium signaling, they will be complemented with the homologous P. japonicum gene. Complementation using phospho-dead and phospho-mimics of these genes will also be performed to determine if the DMBQ-sensitive phosphosite is involved in the DMBQ-responsiveness of the protein. In addition, the promoter of the P. japonicum genes of interest will be used in a reporter gene construct to determine the expression localization of gene of interest in P. japonicum roots. Lastly, the proteins of interest will be expressed in a heterologous system (likely Nicotiana benthamiana) to determine localization and perform any co-immunoprecipitation experiments if appropriate.
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